CN114592070B - Microsatellite family identification method and application of sparus flavescens - Google Patents
Microsatellite family identification method and application of sparus flavescens Download PDFInfo
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Abstract
The invention discloses a microsatellite family identification method for oplegnathus fasciatus, which comprises the following steps: (1) establishing an isotactic cell family of the yellow-fin sea bream; (2) extracting genome DNA of parent and offspring of the yellow-fin sea bream; (3) polymorphic microsatellite primer screening; (4) developing and amplifying a fluorescence labeling 8-fold PCR system; (5) microsatellite locus genotyping and family identification: (5.1) determining the corresponding genotypes of the parent and family individuals at each microsatellite locus; (5.2) family identification. The method establishes a paternity test platform on the yellow-fin snapper by utilizing the fluorescence-modified polymorphic microsatellite marker for the first time, and has high test accuracy, rapidness and low cost. The application of the method in aspects of group genetics evaluation, genealogy authentication, paternity test, molecular marker assisted family management and molecular marker assisted parent selection of the oplegnathus fasciatus is also disclosed.
Description
Technical Field
The invention belongs to the technical field of fish genetic breeding, and particularly relates to a microsatellite family identification method and application of oplegnathus fasciatus.
Background
Yellow sparus (Acanthopagrus latus), also called yellow foot, red wing, shark, huang Qiji sparus. Belonging to the genus Pagrus of the family Pagrus, the class Pagrus, and the genus Pagrus. Oblong, flat and narrow, high, pointed head and forked tail. The dorsal, gluteal, and lower leaves of the caudal are yellow, and there are 11 dorsal fins. The yellow-fin sea bream has strong adaptability and good growth of seawater, salty and fresh water, so the yellow-fin sea bream has an important role in the breeding industry. The distribution of the yellow-fin sea bream extends through the western pacific of india, from the bos bay along the coast of india to the philippines, from australia to japan, and mainly distributes coastal cities such as guangdong, fujian, guangxi and the like in China. The breeding parents of the yellow-fin sea bream are mainly wild catching or cultured population of several generations, the growth speed, disease resistance, other breeding performances and the like of the yellow-fin sea bream do not reach the degree of fine variety improvement yet without artificial breeding, and the adaptability to intensive breeding is weak. Therefore, the development of the fine variety breeding work of the yellow-fin porgy as soon as possible has extremely important significance for protecting the yellow-fin porgy population and meeting the urgent needs of the current healthy breeding industry.
In the research of fish genetic breeding, clear pedigree information is important for family breeding and parent management. In traditional aquatic animal selective breeding, different families need to be maintained by a breeding unit, and the required water is large and inconvenient to manage. In particular, it is considered that there may be some differences in environmental factors between each of the breeding ponds, and that different environmental factors may bias estimates of genetic parameters associated with breeding. At this time, different families can be mixedly cultured together, but all families need to be marked very complexly. In the mixed culture population, family information is kept, and most of livestock researches use physical marks as means, and the physical marks have the limitations of complex operation, certain influence on growth, larger damage to larvae and the like on aquatic animals. The molecular marker makes the identification of the mixed culture kindred relationship possible, and the paternity test technology based on microsatellite typing is one of the most widely and reliably applied means in the current identification of the family of aquatic animals.
Disclosure of Invention
The invention aims to provide a microsatellite family identification method for yellow-fin porgy, which establishes a paternity test platform on yellow-fin porgy by utilizing a fluorescence-modified polymorphic microsatellite marker for the first time, and has the advantages of high identification accuracy, rapidness and low cost.
The invention also aims to provide the application of the method for identifying the microsatellite families of the yellow-fin sea bream in aspects of group genetics evaluation, genealogy authentication, paternity test, molecular marker assisted family management and molecular marker assisted parent selection of the yellow-fin sea bream.
The first object of the present invention can be achieved by the following technical means: a microsatellite family identification method for the oplegnathus fasciatus comprises the following steps:
(1) Establishment of isocytoblast family of yellow-fin sea bream
Artificial propagation is carried out by taking cultured yellow fin sea bream as a parent, an isotactic cell family is established for partial culture, and fries are selected from each family and used as a sample for family identification;
(2) Extraction of genomic DNA of parent and offspring of yellow-fin sea bream
Selecting fin tissues of the parent and the young fish of the yellow-fin sea bream in the step (1), extracting genome DNA of the parent and the offspring, and preserving for later use;
(3) Polymorphic microsatellite primer screening
8 microsatellite loci Y1, Y2, Y3, Y4, Y6, Y8, Y9 and Y10 are obtained by screening from the transcriptome sequencing of the yellow-fin sea bream, 8 pairs of microsatellite primers are designed according to the 8 microsatellite loci, namely a primer pair Y1, a primer pair Y2, a primer pair Y3, a primer pair Y4, a primer pair Y6, a primer pair Y8, a primer pair Y9 and a primer pair Y10;
(4) Fluorescent-labeled 8-fold PCR system development and amplification
Designing 8 pairs of primers screened in the step (3) into an 8-fold PCR system, respectively modifying the 5' end of the forward primer of the 8 pairs of primers by using three different fluorescent groups of FAM (blue), HEX (green) and TAMRA (pink), and carrying out PCR amplification on the DNA samples of the parents and the offspring in the step (2) by adopting a fluorescent PCR reaction;
(5) Microsatellite locus genotyping and family identification
(5.1) determining the corresponding genotypes of the parent and family individuals at each microsatellite locus
Typing the 8-fold PCR amplification product in the step (4) on a sequencer;
(5.2) family identification
Analyzing the parent genotype and the offspring genotype, and judging the parent and the child of the offspring individual.
In the method for identifying the microsatellite family of the yellow sparus praecox:
preferably, in the step (2), the genomic DNA of the parent and the offspring is extracted by an ammonium acetate method.
Preferably, in the step (3), the base sequences of the primer Y1, the primer pair Y2, the primer pair Y3, the primer pair Y4, the primer pair Y6, the primer pair Y8, the primer pair Y9 and the primer pair Y10 are respectively shown in SEQ ID NOs: 1 to 16.
Preferably, the 8-fold PCR system in step (4) comprises: 10. Mu. Mol/L primer set Y1.5. Mu. L, 10. Mu. Mol/L primer set Y2.3. Mu. L, 10. Mu. Mol/L primer set Y3.4. Mu. L, 10. Mu. Mol/L primer set Y4.5. Mu. L, 5. Mu. Mol/L primer set Y6.5. Mu. L, 5. Mu. Mol/L primer set Y8.6. Mu. L, 5. Mu. Mol/L primer set Y9.6. Mu. Mol/L, 5. Mu. Mol/L primer set Y10.4. Mu. L, 100 ng/. Mu. L DNA 3. Mu. L, ddH) 2 O 6.2μL、PCR Starmix 12. Mu.L, 25. Mu.L total.
Preferably, in the step (4), the fluorescent substance FAM is labeled at the 5' end of the forward primer of the primer pair Y1, the primer pair Y2, and the primer pair Y3, the fluorescent substance HEX is labeled at the 5' end of the forward primer of the primer pair Y4, and the primer pair Y6, and the fluorescent substance TAMRA is labeled at the 5' end of the forward primer of the primer pair Y8, the primer pair Y9, and the primer pair Y10.
Preferably, in the PCR amplification in the step (4), the PCR reaction procedure is as follows: pre-denaturation at 94℃for 5min, then annealing at 94℃for 30s, annealing at 59℃for 30s, and annealing at 72℃for 30s, for a total of 30 cycles, and finally extension at 72℃for 10min.
Preferably, in step (5.1), the 8-fold PCR amplification product of step (4) is typed on an ABI3730XL gene analyzer, and the genotype of each sample is read using the GeneMarker V1.5 software using GS-500 as an internal reference.
Preferably, in the step (5.2), the parent genotype and the offspring genotype are analyzed by using CERVUS 3.0 software, the parent and the offspring individual parent are judged, the result is compared with the known actual family information, and finally the success of family identification is judged.
Preferably, in step (5.2) the parental genotype and the offspring genotype are analyzed using software CERVUS 3.0, the software CERVUS 3.0 is used to calculate the allele frequency, heterozygosity, desired heterozygosity, polymorphic information content, average exclusion probability, hardy-Weinberg equilibrium and null allele frequency of the parental genotype and offspring genotype at each microsatellite locus, and identify the parent and parent of each family individual based on the LOD value.
The second object of the present invention can be achieved by the following technical means: the method is applied to group genetics evaluation, genealogy authentication, paternity test, molecular marker assisted family management and molecular marker assisted parent selection of the oplegnathus fasciatus.
Compared with the prior art, the invention has the following advantages:
(1) The invention utilizes the combination of microsatellite markers and multiple fluorescent PCR technology to screen 8 high polymorphism microsatellite loci, designs 8 pairs of primers, constructs an 8-fold fluorescent PCR system, and performs individual identification and parent relationship identification on the yellow fin sea bream family by parting with a sequencer;
(2) The invention can detect 8 sites at a time, and compared with simple single-site detection, the invention has the advantages of improved efficiency and reduced cost by about one eighth of the original cost;
(3) The invention can be applied to identification of the mixed culture family of the sparus praecox, and the offspring of each family does not need to be fed separately in production, so that the water body and the manual management are saved, the cost is reduced, and meanwhile, the error caused by environmental factors is overcome, so that the paternity test technology can be widely popularized in reproduction;
(4) The microsatellite loci selected by the invention have more alleles and high polymorphism, can be used for group genetic evaluation, genealogy authentication and paternity test of the oplegnathus fasciatus, and can also be used for molecular marker assisted family management and molecular marker assisted parent selection.
Detailed Description
The present invention will be described in further detail by way of examples, but embodiments of the present invention are not limited thereto.
Example 1
The microsatellite family identification method for the sparus flavescens provided by the embodiment comprises the following steps:
(1) Establishment of yellow-fin sea bream family
10 parents of the red sea bream are collected from Yangjiang and Daya, artificial induced spawning propagation is carried out, the ratio of male to female is 1:1, and 10 isotactic cell families are established. And cutting fin tissues of each family parent, putting the fin tissues into absolute ethyl alcohol, recording family information, and preserving at-20 ℃ for later use. 10 families are put in a circulating water system for separate cultivation, 10 fish are randomly selected from each family after the fries hatch for 45 days, and the 10 fish are fixed by absolute ethyl alcohol and used as a sample for family identification.
(2) Extracting genome DNA of parent and offspring of the yellow-fin sea bream;
the fin tissues of the parent and offspring are placed in 2mL centrifuge tubes respectively, the tissues are sheared by scissors, 600. Mu.L of cell lysate (Tris-HCl 100mM,pH 8.0;EDTA 50mm/L, pH 8.0; SDS 1%, pH 8.0; naCl 125 mM) is added, proteinase K9. Mu.L with the concentration of 20mg/mL is added into each tube, the tubes are placed in a water bath kettle with the temperature of 60 ℃ for 2-4h, and the centrifuge tubes are shaken every half an hour until the tissues are fully lysed. The tube was cooled to room temperature, 200. Mu.L of 7.5M ammonium acetate was added, shaken well, placed in a refrigerator at 4℃for 5min, centrifuged at 12,000rpm at 4℃for 10min, and 500mL of supernatant was taken to a new 1.5mL centrifuge tube. 600mL of isopropanol was added, mixed well, precipitated at room temperature for 1-2min, centrifuged at 12,000rpm at 4℃for 10min, and the isopropanol was discarded. The DNA was washed with 70% alcohol, centrifuged at 12,000rpm at 4℃for 5min, and 70% alcohol was discarded. Adding absolute ethanol, centrifuging at 12,000rpm and 4deg.C for 5min, discarding absolute ethanol, repeating for several times, drying at room temperature for about 30min, and adding 100 μl of double distilled water to dissolve DNA. The DNA concentration and quality were measured with a NanoDrop ND-1000 UV spectrophotometer and each DNA sample was diluted to 100 ng/. Mu.L.
(3) Polymorphic microsatellite marker screening:
8 microsatellite loci Y1, Y2, Y3, Y4, Y6, Y8, Y9 and Y10 obtained from high-throughput sequencing analysis of the yellow-fin sea bream are utilized to amplify in 30 yellow-fin sea bream individuals, and finally 8 pairs of primers with clear bands and high polymorphism are selected as primers for family identification, so that an 8-fold PCR system is designed, and the 8 pairs of microsatellite primers are respectively: primer pair Y1, primer pair Y2, primer pair Y3, primer pair Y4, primer pair Y6, primer pair Y8, primer pair Y9, and primer pair Y10.
The 5' end of the forward primer of the 8 pairs of microsatellite primers is modified by using FAM, HEX, TAMRA three different fluorescent groups respectively, the amplified fragments of the same fluorescent modified primers have different size ranges, and the specific fragment sizes are shown in the following table 1,
TABLE 1 primer sequences and fluorescent substances for 8-PCR combination of Pagrus major
Note that: f represents the forward primer, R represents the reverse primer, and all fluorescent substances are marked on the 5' end of the forward primer.
(4) Development and amplification of 8-fold PCR conditions
Combining the 8 pairs of primers selected above into an 8-fold PCR, wherein the 5' -end of the forward primer of each pair of primers is marked with a fluorescent substance, and the method comprises the following steps: primer pair Y1, primer pair Y2, primer pair Y3 labeled fluorescent material FAM (blue); primer pair Y4 and primer pair Y6 are marked with fluorescent substance HEX (green); primer pair Y8, primer pair Y9 and primer pair Y10 label fluorescent material TAMRA (pink). The total system of the 8-fold PCR system is shown in Table 2:
TABLE 2 paternity test PCR reaction System of yellow-fin sea bream
Setting a PCR reaction program: pre-denaturation at 94℃for 5min, then annealing at 94℃for 30s, annealing at 59℃for 30s, and annealing at 72℃for 30s, for a total of 30 cycles, and finally extension at 72℃for 10min. After the PCR was completed, 5. Mu.L of the sample was subjected to electrophoresis on agarose gel, and the sample was sent to commercial company for genotyping using ABI3730 XL.
(5) Microsatellite locus genotyping and paternity analysis
The amplified products were typed on an ABI3730XL gene analyzer using GS-500 as an internal control, the genotype of the individual was read using GeneMarker V1.5 software, the allele frequency, heterozygosity, expected heterozygosity, polymorphic information content, mean exclusion probability, hardy-Weinberg balance and null allele frequency of the parents and offspring at each microsatellite locus were calculated using software cerus 3.0, and the parental identity of each family individual was identified based on LOD values (see table 3).
TABLE 3 statistical and exclusion probabilities for genetic diversity of 8 microsatellite loci
| Primer name | k | HO | HE | PIC | Excl 1 | Excl 2 | HW | F(Null) |
| Y1 | 20 | 0.916 | 0.915 | 0.890 | 0.338 | 0.281 | NS | -0.0043 |
| Y2 | 14 | 0.894 | 0.893 | 0.852 | 0.422 | 0.266 | NS | -0.0215 |
| Y3 | 13 | 0.892 | 0.891 | 0.870 | 0.385 | 0.235 | NS | -0.0158 |
| Y4 | 17 | 0.908 | 0.906 | 0.884 | 0.366 | 0.217 | NS | +0.0079 |
| Y6 | 10 | 0.842 | 0.841 | 0.849 | 0.321 | 0.269 | NS | +0.0074 |
| Y8 | 16 | 0.905 | 0.902 | 0.867 | 0.389 | 0.247 | NS | +0.0289 |
| Y9 | 12 | 0.890 | 0.888 | 0.866 | 0.377 | 0.231 | NS | +0.0258 |
| Y10 | 14 | 0.894 | 0.892 | 0.888 | 0.378 | 0.239 | NS | +0.0278 |
Note that: k is the number of alleles, ho is the observed heterozygosity, HE is the desired heterozygosity, PIC is polymorphic content, excl 1 is the exclusion rate when the parent is unknown, excl 2 is the exclusion rate when the parent is known, HW is the hadowberg equilibrium test, NS indicates insignificant deviation, and F (Null) indicates Null allele frequency.
(5) Family identification result
In the simulation analysis using the CERVUS 3.0, 10000 filial generations are generated by using 10 pairs of parents for simulation, the success rate of paternity test can reach 100% in the range of 80% and 95% confidence intervals, and in 100 individuals of 10 families which are actually tested, 1 case does not find the true female parent and male parent, and mismatch occurs. The probability of finding the true parent from the candidate parents is 99%, so that the requirements of genealogy analysis and family management in genetic breeding can be met.
The results show that the microsatellite 8-heavy fluorescence PCR method is stable and accurate in parent-child identification of the garrupa line, and can meet the requirements of garrupa line germplasm identification, family management and proliferation and release effect evaluation.
The invention has been described with reference to a few specific embodiments, it being necessary to note that the above specific embodiments are provided for the purpose of further illustration and are not intended to limit the scope of the invention. Some insubstantial modifications and adaptations of the invention by others are within the scope of the invention.
Claims (5)
1. A microsatellite family identification method for oplegnathus fasciatus is characterized by comprising the following steps:
(1) Establishment of isocytoblast family of yellow-fin sea bream
Artificial propagation is carried out by taking cultured yellow fin sea bream as a parent, an isotactic cell family is established for partial culture, and fries are selected from each family and used as a sample for family identification;
(2) Extraction of genomic DNA of parent and offspring of yellow-fin sea bream
Selecting fin tissues of the parent and the young fish of the yellow-fin sea bream in the step (1), extracting genome DNA of the parent and the offspring, and preserving for later use;
(3) Polymorphic microsatellite primer screening
8 microsatellite loci Y1, Y2, Y3, Y4, Y6, Y8, Y9 and Y10 are obtained by screening from the transcriptome sequencing of the yellow-fin sea bream, 8 pairs of microsatellite primers are designed according to the 8 microsatellite loci, namely a primer pair Y1, a primer pair Y2, a primer pair Y3, a primer pair Y4, a primer pair Y6, a primer pair Y8, a primer pair Y9 and a primer pair Y10;
the sequences of the forward and reverse primers of primer pair Y1 are as follows:
F：TGGTTGCTGACGTATCCTGG；
R：TGTTGGTTTCTGTCCGTGGA；
the sequences of the forward and reverse primers of primer pair Y2 are as follows:
F：TACTGCCTGGTGTGAAAGCC；
R：CCCACCCATTTAGCTGGAGG；
the sequences of the forward and reverse primers of primer pair Y3 are as follows:
F: AGCTGAGACCCTCTGAGGAG；
R: GGTACCTCTGACTGAGCTGC；
the sequences of the forward and reverse primers of primer pair Y4 are as follows:
F: ACACACGCCTAAACACACCA；
R: ATCACAGCGTCTCCCTCTCT；
the sequences of the forward and reverse primers of primer pair Y6 are as follows:
F: AGTTCAGGCAGCAGGTTGTT；
R: GAGTCATCAGGAGCAGGACG；
the sequences of the forward and reverse primers of primer pair Y8 are as follows:
F: GAAGCGGAGTCTGGTGGAAA；
R: CTGAATCCCTCAGCCACCTC；
the sequences of the forward and reverse primers of primer pair Y9 are as follows:
F: GGACTGCTCCTGTTCCTGTC；
R: TGGGGGCAGACAGATAGACA；
the sequences of the forward and reverse primers of primer pair Y10 are as follows:
F: TGGTTCGGTTTCCTGTCCTG；
R: TGCCTCATGGTGAGTTCAGG；
(4) Fluorescent-labeled 8-fold PCR system development and amplification
Designing 8 pairs of primers screened in the step (3) into an 8-fold PCR system, respectively modifying 5' ends of forward primers of the 8 pairs of primers by using FAM, HEX, TAMRA different fluorescent groups, and carrying out PCR amplification on the parent and offspring DNA samples in the step (2) by adopting a fluorescent PCR reaction;
in the step (4), fluorescent materials FAM are marked at the 5' ends of the forward primers of the primer pair Y1, the primer pair Y2 and the primer pair Y3, fluorescent materials HEX are marked at the 5' ends of the forward primers of the primer pair Y4 and the primer pair Y6, and fluorescent materials TAMRA are marked at the 5' ends of the forward primers of the primer pair Y8, the primer pair Y9 and the primer pair Y10;
(5) Microsatellite locus genotyping and family identification
(5.1) determining the corresponding genotypes of the parent and family individuals at each microsatellite locus
Typing the 8-fold PCR amplification product in the step (4) on a sequencer;
(5.2) family identification
Analyzing the parent genotype and the offspring genotype, and judging the parent and the child of the offspring individual.
2. The method for identifying the microsatellite family of the yellow fin sea bream according to claim 1, which is characterized in that: and (3) extracting genome DNA of the parents and the filial generations by adopting an ammonium acetate method in the step (2).
3. The method for identifying the microsatellite family of the yellow fin sea bream according to claim 1, which is characterized in that: the 8-fold PCR system in the step (4) comprises: primer pair Y1.5 [ mu ] mol/L, primer pair Y2.3 [ mu ] L, primer pair Y3.4 [ mu ] L, primer pair Y4.5 [ mu ] L, primer pair Y6.5 [ mu ] L, primer pair Y8.6 [ mu ] L, primer pair Y9.6 [ mu ] L, primer pair Y10.4 [ mu ] L, and DNA 3 [ mu ] L, ddH, wherein the concentration of the primer pair Y1.5 [ mu ] mol/L, the concentration of the primer pair Y2.3 [ mu ] L, the concentration of the primer pair Y10.4 [ mu ] L, the concentration of the primer pair Y4.5 [ mu ] mol/L, the concentration of the primer pair Y6.5 [ mu ] mol/L, the concentration of the primer pair Y8.6 [ mu ] L, the primer pair Y9.6 [ mu ] L, the concentration of the primer pair Y9.6 [ mu ] L, the primer pair Y10.4 [ mu ] L, and the concentration of the primer pair Y3 [ mu ] L, ddH, and the primer pair 3 [ mu ] L 2 O6.2mu. L, genStar, taq PCR Starmix 12 mu L and total 25 mu L.
4. The method for identifying the microsatellite family of the yellow fin sea bream according to claim 1, which is characterized in that: in the step (4), during PCR amplification, the PCR reaction procedure is as follows: pre-denaturation at 94℃for 5min, then annealing at 94℃for 30s, annealing at 59℃for 30s, and annealing at 72℃for 30s, for a total of 30 cycles, and finally extension at 72℃for 10min.
5. The use of the method of claim 1 in paternity testing and molecular marker assisted parent selection of oplegnathus fasciatus.
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| CN116555433B (en) * | 2022-12-28 | 2024-03-19 | 中国水产科学研究院南海水产研究所 | Primer and kit for identifying Huang Qiji and flat sea bream and application |
| CN116179657B (en) * | 2022-12-30 | 2023-09-05 | 中国水产科学研究院珠江水产研究所 | Primer combination, microsatellite marker combination, multiplex PCR system, method for identifying snakehead, and application of multiplex PCR system |
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| CN109055571A (en) * | 2018-08-28 | 2018-12-21 | 中国水产科学研究院南海水产研究所 | The specific primer of yellowfin spine porgy microsatellite marker and application |
| CN113667760A (en) * | 2021-07-06 | 2021-11-19 | 中山大学 | SSR marker primer and method for evaluating genetic diversity of sparus latus population |
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| CN109055571A (en) * | 2018-08-28 | 2018-12-21 | 中国水产科学研究院南海水产研究所 | The specific primer of yellowfin spine porgy microsatellite marker and application |
| CN113667760A (en) * | 2021-07-06 | 2021-11-19 | 中山大学 | SSR marker primer and method for evaluating genetic diversity of sparus latus population |
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